Incubation of 32P-labeled rat adipocytes with insulin induced serine phosphorylation and activation of the cGMP-inhibited, low Km cAMP PDE (CGI PDE). To study further CGI PDE regulation, effects of phorbol ester (PMA) and Ca2+-mobilizing hormones were compared to insulin. Insulin evoked 4-30-fold and approximately 50% increases in CGI PDE phosphorylation and activation, respectively; the corresponding effects of PMA, vasopressin and angiotensin II were approximately 1/4 as large. When present with insulin, PMA or vasopressin inhibited (30-50%) its effects. On SDS-PAGE autoradiographs, V8 protease generated several peptides from 32P-CGI PDE isolated from either insulin- or PMA-treated cells; a approximately 24 kDa peptide was unique to PMA. At least four peptides with Rf values ranging from 0.08-0.39 (on thin layer chromatography) were generated by trypsin and chymotrypsin digestion of 32P-CGI PDE from cells incubated with insulin; only the lowest mobility peptide was observed with PMA, and treatment of cells with both insulin and PMA diminished insulin-specific 32P-peptides. These results suggest that adipocyte CGI PDE is activated by insulin and cAMP, as well as PMA and Ca2+mobilizing hormones. Although PMA and Ca2+-mobilizing hormones evoke small increases in phosphorylation and activation of CGI PDE, Ca2+-dependent regulation appears to interfere with, rather than completely mimic the effects of insulin, perhaps via competing inhibitory phosphorylation site(s). To identify the insulin-stimulated serine kinase(s) which regulates CGI PDE, a cell-free assay system was developed. A time-, concentration- and ATP-dependent phosphorylation of particulate rat adipocyte CGI PDE was observed when membranes were incubated with cytosol prepared from insulin-treated, but not control, cells. Extracts from insulin-treated cells increased CGI PDE phosphorylation approximately 4-fold. Pure cAMP-dependent protein kinase catalytic subunit phosphorylated CGI PDE in vitro. However, the insulin-stimulated factor(s) is cAMP-independent, and different from cAMP-dependent protein kinase, casein kinase II, various S6-kinases, protease-activated kinase II or protein kinase C.